Improvement of subtilisin-like serine alkaline protease by directed evolution for cold-adaptation
The activities in low temperatures of a mesophilic alkaline protease TC4 was improved by directed evolution. The process involved random mutagenesis by error-prone PCR and DNA shuffling followed by first screening on skim-milk plate and second screening by 96-well plate with AAPF as the substrate. It yielded a mutant T31, with a kcat 5 times and a catalytic efficiency kcat/Km 6.33 times greater than that of wild type TC4 at 20 ℃. The relative activity of T31 at 20 ℃ was improved as much as 25% compared to that of wild type enzyme TC4 with casein as the substrate. Four amino acid substitutions were found in T31 and the results showed that these amino acid substitutions were sufficient to generate a mutant whose low temperature activity was greater than that of TC4.
KunCheng Fuping Lu Ming Li Xiaomei Liang
Educational Bureau Key Laboratory of Industrial Microorganism, Institute of Bioengineering Tianjin University of Science and Technology Tianjin, China
国际会议
成都
英文
1-4
2010-06-18（万方平台首次上网日期，不代表论文的发表时间）